Prior to the present invention, methods for determining ions in solution included flame photometry, atomic absorption photometry, ion-selective electrodes, multiple liquid phase partitioning and colorimetric slides. The use of certain compounds and compositions which selectively complex with, and therefore isolate, certain ions from the sample solution has become popular in ion-selective electrodes. These substances, known as ionophores, have the capability of selectively isolating ions from their counterions and other ions in a test sample, thereby causing a charge separation and a corresponding change in electrical conductivity in the phase containing the ionophore. Illustrative of other uses of the ion/ionophore phenomenon include ion assays utilizing membrane electrodes, liquid/liquid partitioning, fluorescence, various reporter substances, and chromogenic derivatives of certain ionophoric compounds.
2.1 Ion-Selective Electrodes (ISE)
When two solutions having different concentrations of ions are separated by an electrically conductive membrane, an electromotive force (EMF) can be generated. The EMF developed by such a system is a function of concentration or ionic activity of the solutions on either side of the membrane. This phenomenon is expressed mathematically by the well-known Nernst Equation ##EQU1## in which E is the EMF of the particular system, F is the Faraday Constant, R is the gas constant, T is the temperature in .degree.K and .gamma. and c are, respectively, the activity coefficients and molal concentrations of the ion under study. The subscript 1 designates the solution on one side of the membrane; the subscript 2 denotes the solution on the other side. The charge of the ion involved in the reaction is denoted by n.
In such membrane separation cells, the membrane can be a simple fritted glass barrier, allowing a small but measurable degree of ion diffusion from one solution to the other. Alternatively, a nonporous, electrically nonconductive film, such as polyvinyl chloride, impregnated with an ionophore can be employed. In the absence of the ionophore the film is an insulator and no EMF can be measured; when blended with an ionophore, charged ions are bound to the film and a small, measurable current can be induced to flow. Because the ionophore is selective in its affinity, and thus will bind only certain specific ions, such cells are ion selective. Any measurable EMF is due solely to the presence of those ions.
It is known that certain antibiotics, such as valinomycin, have an effect on the electrical properties of phospholipid bilayer membranes (biological membranes), such that these antibiotics effect solubilization of cations within the membrane, in the form of mobile charged complexes, thereby providing a "carrier" mechanism by which cations can cross the insulating hydrophobic or hydrocarbon interior of the membrane. Such complexes have the sole purpose of carrying the charge of the complex through the membrane In an ISE they cause a voltage differential which can be determined between solutions on either side of the ISE membrane.
Thus, a cell for determining potassium ion can be produced through use of an ionophore specific for potassium (K.sup.+), e.g. valinomycin. In the presence of K.sup.+, valinomycin produces a concentration gradient across a membrane by binding and transporting the ion, thus generating a potential across the membrane. A reference concentration of K.sup.+ is placed on one side of the membrane and the test sample on the other. The EMF developed is measured using external reference electrodes and used to calculate the unknown concentration from equation (1). Because only K.sup.+ binds to the valinomycin in the membrane, the conductive path only appears for K.sup.+. Therefore, the EMF developed is attributable solely to the K.sup.+ concentration gradient across the membrane.
The current flowing across the membrane is so small that no significant quantity of K.sup.+ or counterion is transported through it. Electrical neutrality of the membrane is maintained either by a reverse flow of hydrogen ions (protons), or by a parallel flow of OH.sup.-.
A major difficulty in the use of such ion-selective electrodes has been the marked reduction of accuracy, selectivity and speed of response over time. Further, small changes in ion concentration produce such small changes in EMF that sophisticated voltmeter equipment is required.
Swiss Patent Application Serial No. 11428/66, filed Aug. 9, 1966, describes the use of porous membranes impregnated with macrocyclic derivatives of amino and oxy-acids in ion-sensitive electrodes. Materials used to form the membrane are glass frits and other porous membranes. Such electrodes are said to be effective in measuring ion activities.
U.S. Pat. No. 4,053,381, issued to Hamblen, et al., discloses similar technology, and utilizes an ion specific membrane having ion mobility across it.
2.2 Liquid/Liquid Partitioning
Another known application of ionophores in ion determination is through liquid/liquid partitioning. Eisenman et al., J. Membrane Biol., 1, 294-345 (1969), disclose the selective extraction of cations from aqueous solutions into organic solvents via macrotetralide actin antibiotics. In this procedure, a hydrophobic ionophore is dissolved in an organic solvent immiscible with water. The technique involves shaking an organic solvent phase containing the antibiotics with aqueous solutions containing cationic salts of lipid-soluble colored anions, such as picrates and dinitrophenolates. The intensity of color of the organic phase is then measured spectrophotometrically to indicate how much salt has been extracted. Phase transfer has also been studied by Dix et al., Angew, Chem. Int. Ed. Engl., 17, 857 (1978) and is reported in reviews including Burgermeister et al., Top. Curr. Chem., 69, 91 (1977); Yu et al., "Membrane Active Complexones," Elsevier, Amsterdam (1974); and Duncan, "Calcium in Biological Systems," Cambridge University Press (1976).
Sumiyoshi, et al., Talanta, 24, 763-5 (1977) describe another method useful for determining K.sup.+ in serum. In this technique serum is deproteinated by trichloroacetic acid, an indicator dye is added, and the mixture shaken with a solvent such as chloroform containing valinomycin.
Partitioning of a compound is rapid and effective between liquids, as shown by Eisenman, because of the mobility of the ionophore carrier and ions in their respective phases, which allows the transported species to diffuse rapidly away from the interface. Such a mechanism is normally impossible in the solid phase, because of the rigidity, immobility and essentially zero diffusion of materials in a solid phase.
2.3 Fluorescent Anions
Yet another approach to the measurement of ion activity in aqueous solutions utilizes fluorescent anions. Feinstein, et al., Proc. Nat. Acad. Sci. U.S.A., 68, 2037-2041 (1971). It is stated that the presence of cation/ionophore complexes in organic solvents is known, but that complex formation in purely aqueous media had theretofore not been detected. Feinstein, et al., demonstrated the existence of such complexes in water through the use of the fluorescent salts 1-anilino-8-naphthalene sulfonate and 2-p-toluidinyl sulfonate.
It was found that interaction of the ionophore/cation complex with the fluorescent dyes produced enhanced fluorescence emission, increased lifetime and polarization, and significant blue-shift at the emission maxima of the fluorescence spectra. At constant concentrations of ionophore and fluorophore, the intensity of fluorescence emission was found to be a function of cation concentration.
2.4 Reporter Substances
As indicated supra, anionic dyes and fluorescers can be induced to enter the organic phase of a two-phase liquid system by the presence in that phase of a cation/ionophore complex. Thus these detectable anions can be said to "report" the presence of the cation trapped by the ionophore in the organic phase.
Other reporter substances which are not ionic in nature can be induced by the ionophore/cation complex to undergo a reaction yielding a detectable product An example is the reaction sequence reported in U.S. Pat. No. 4,540,520 whereby a cation/ionophore complex induces a phenol to become deprotonated, thus initiating a coupling reaction to form a colored product. The so-called Gibbs Reaction is typical of such a reporter substance-producing reaction, in which 2,5-cyclohexadiene-1-one-2,6-dichloro-4-chloroimine couples with a deprotonated phenol to form a colored product and HCl.
2.5 Ionophores
The term "ionophore" embraces many diverse molecules, all of which are related by their unique capacity to bind with certain charged species to the relative exclusion of others, and which do so in a fashion which, at least to some degree, enables the ionophore molecule to electrically shield the ion from its environment. Indicative of this phenomenon is the liquid/liquid partitioning technique described above. The ionophore, because of its unique structure and its multitude of electron rich or electron deficient atoms ("donor atoms" or "receptor atoms", respectively) enables an ion such as sodium or potassium to enter a nonpolar organic phase.
Ionophores include naturally occurring compounds, such as valinomycin, as well as compounds of the structural categories of podands, corands, cryptands, hemispherands, spherands and cryptahemispherands.
2.5.1 Podands
Ions can be selectively complexed with certain acyclic compounds. For example, a linear chain which contains a regular sequence of electron rich donor atoms, such as oxygen, sulfur or nitrogen, has the capability of associating with positively charged ions to form complexes. The main structural difference between podands and other ionophores is the openness or acyclic nature of their structures. Thus podands can be subcategorized into monopodands, dipodands, tripodands, etc. A monopodand, therefore, is a single organic chain containing donor or receptor atoms, a dipodand is two such chains connected to a central moiety capable of variable spacial orientation, and a tripodand is three chains attached to a central moiety.
2.5.2 Corands
The corands are monocyclic compounds which contain electron donor atoms or acceptor atoms, which are electron rich or deficient, and which are capable of complexing with particular cations or anions because of their unique structures. Included in this term are the crown ethers in which the monocyclic ring contains oxygen as the donor atoms. Other corands are compounds which contain an assortment of electron rich atoms such as oxygen, sulfur and nitrogen. Because of the unique sizes and geometries of particular corands, they are adaptable to complexing with various ions. In so complexing, the electron rich atoms, such as the oxygens in a crown ether, become spacially oriented towards the electron deficient cation. The carbon atom segments of the chain are simultaneously projected in a direction outwards from the ion. Thus, the resultant complex is charged in the center but is relatively hydrophobic at its perimeter.
2.5.3 Cryptands
The cryptands are the polycyclic analogs of the corands. Accordingly, they include bicyclic and tricyclic multidentate compounds. In the cryptands, the cyclic arrangement of donor atoms is three dimensional in space, as opposed to the substantially planar configuration of the corand. A cryptand is capable of virtually enveloping the ion in three dimensional fashion and, hence, is capable of strong bonds to the ion in forming the complex. As with the corands, the donor atoms can include such atoms as oxygen, nitrogen and sulfur.
2.5.4 Hemispherands
Hemispherands are macrocyclic or macropolycyclic ionophore systems, such as cryptands, whose cavities are partially preorganized for binding by the rigidity of the hydrocarbon support structure and the spatial and orientational dictates of appended groups.
2.5.5 Spherands
Spherands are macrocyclic or macropolycyclic ionophore systems whose cavities are fully preorganized by their synthesis, as opposed to becoming organized during complexing such as with an ion.
2.5.6 Cryptahemispherands
Cryptahemispherands combine the partially preorganized cavity features of the hemispherand, but contain multiple other ligand-gathering features of the cryptands.
2.6 Chromogenic Ionophores
Certain compounds have been studied which are capable not only of behaving as ionophores by forming cation complexes but which, when so complexed, exhibit a detectable formation of or change in color. Thus, experiments were published in 1977 whereby chromophoric moieties were covalently attached to ionophores to achieve a color response to potassium (Tagaki, et al., Analytical Letters, 10 (13), pp. 1115-1122 (1977)). There it is taught to couple covalently a chromophoric moiety such as 4-picryl-amino- to an ionophore such as benzo15-crown-5. Moreover, U.S. Pat. No, 4,367,072 mentions many crown ethers, cryptands and podands covalently substituted with a chromophoric group, such as ##STR3## Yet another reference, German Offenlegungschrift 32 02 779, published Aug. 4, 1983 discloses a chromogenic cryptand structure.
2.7 Synopsis
Many technological developments have occurred since the early recognition that antibiotics such as valinomycin are capable of complexing certain ions and transporting them into the hydrophobic internal region of a cell membrane and, ultimately, into the cell nucleus. This basic ionophore discovery has led to the invention of a myriad of assay techniques for such ions as potassium, sodium, calcium and others; and has spawned a variety of diagnostic procedures of invaluable assistance to the chemist and physician. Moreover, countless new ionophore compounds have been discovered and invented of such chemical and structural diversity and complexity as to engender a whole new area of organic chemistry.
Certain applications of these technologies to ion determination, however, have met with problems. Although ionophores can possess high ion selectivity, the presence of high concentrations of other ions relative to the ion of interest can lead to interference in the desired result. Thus, if an ionophore were to have a specificity ratio of 50:1 for complexing with ion X.sup.+ over ion Y.sup.+, nevertheless if Y.sup.+ were present in solution at a concentration 50 times that of X.sup.+, the resultant selectivity of the system for X.sup.+ would be diminished to such a great extent as to render the ionophore practically useless as an assay reagent for X.sup.+. Such disparity of concentrations occurs, for example, in blood where normal sodium/potassium concentration ratios are in the neighborhood of 35:1.
Moreover, some prior art assays utilizing prior art ionophores have heretofore required a highly alkaline medium in order to function usefully, and aspects which contribute to poor shelf life as well as corrosiveness. Such systems also require a hydrophobic phase to contain or segregate the ionophore from the aqueous test sample, thus leading to organic/aqueous systems which respond relatively slowly.
Thus, it would be desirable to greatly increase selectivity in a chromogenic ionophore, thereby overcoming interference from competing ions present at much higher concentrations. Likewise, it would be desirable to obviate the need for harshly alkaline conditions and a multiphasic system. These and other unexpected advantages have been realized through utilizing the unique compounds described herein.